Structural basis of S-adenosylmethionine-dependent allosteric transition from active to inactive states in methylenetetrahydrofolate reductase

Methylenetetrahydrofolate reductase (MTHFR) is a pivotal flavoprotein connecting the folate and methionine methyl cycles, catalyzing the conversion of methylenetetrahydrofolate to methyltetrahydrofolate. Human MTHFR (hMTHFR) undergoes elaborate allosteric regulation involving protein phosphorylation and S-adenosylmethionine (AdoMet)-dependent inhibition, though other factors such as subunit orientation and FAD status remain understudied due to the lack of a functional structural model. Here, we report crystal structures of Chaetomium thermophilum MTHFR (cMTHFR) in both active (R) and inhibited (T) states. We reveal FAD occlusion by Tyr361 in the T-state, which prevents substrate interaction. Remarkably, the inhibited form of cMTHFR accommodates two AdoMet molecules per subunit. In addition, we conducted a detailed investigation of the phosphorylation sites in hMTHFR, three of which were previously unidentified. Based on the structural framework provided by our cMTHFR model, we propose a possible mechanism to explain the allosteric structural transition of MTHFR, including the impact of phosphorylation on AdoMet-dependent inhibition.

The text and experiments about phosphorylation (lines 102-130) should be mostly removed as phosphorylation is not addressed in this manuscript.Move it to the Supplementary Information (SI).
The main text should simply mention the authors' attempt to crystallize the human enzyme and the discovery of new phosphorylation sites.
Line 131 should explicitly state that the Arg357Cys mutation was chosen based on published data.
In Figure 2 and associated text, provide steady-state kcat and KM parameters for the fungal enzyme.The Figure 2 legend should list the enzyme and substrate concentrations used to obtain the curves and spectra shown.
In Figure 3, the legend should indicate which ligands are bound and the coloring used to depict them.
Lines 200-215: Move the limited proteolysis data text after presenting the 3D structure.Use it as an experiment validating the relevance in solution of the described conformational changes.
Line 230: The biochemical properties of Arg315Ala should be minimally described (activity, spectra).
The methods indicate that R-state (not the T-state?) crystallisation was done with a triple E21Q, L393M, V516F mutant where the lysines were vhemically methylated.Please explain this in the main text.
Figure 5 is confusing due to a likely mistake in residue numbering.For instance, the legend states that Arg357 interacts with Glu360, whereas the figures outline an interaction between Arg358 and Glu360.Thr368 is not sohwin in the figure .Related to the above point: The manuscript is sometimes challenging to follow due to the backand-forth between the residue numbering of the human and fungal enzyme (e.g., lines 290-300).I recommend using the residue numbering of the fungal enzyme for consistency.

Reviewer #2 (Remarks to the Author):
The following paper reports on the structures of R-state and T-state (inhibited) form of MTHFR from the fungus Chaetomium thermophilum.cMTHFR serves as a suitable model for understanding the multifaceted allosteric regulation of mammalian MTHFR, and important enzyme that links one carbon folate and methionine metabolism.The work is significant because (i) it provides a structural rationale for how 2 molecules of SAM act as allosteric inhibitors and (ii) reveals a new mechanism for allosteric control of an enzyme, namely the interweaving of mobile linker region between the regulatory and catalytic domains of the enzyme.The structures also lead to a proposal for how phosphorylated of residues at the N-terminus contributes to stabilization of the T-state.The work also supports or explains published biochemical/biophysical data for MTHFR, for example perturbation of the FAD absorbance spectra induced by SAM.
The poor atomic resolution of both the R-and T-state of the cMTHFR structures is a limitation of the study.The resolution is suitable for tracking the reorganization of domain, but perhaps not for mapping intermolecular interactions.Can you show an H-bond between R358 and Glu360 at 3.4 A resolution?
Biochemical data supporting the binding of two equivalent of SAM would confirm that the second binding site is physiologically relevant or at least happens in solution.Can this be done with ITC?Given that site 2 is occluded in the R state and site 1 is not, they would presumably have different binding affinities.You could measure SAM binding to wild type cMTHFR and the R315 mutant.Did you subject the R315C variant to limited proteolysis (with and without SAM).This would support its stabilization of the T-state.
Line 227; Abolishment of activity (?) or a significantly reduced activity.The R315C still shows activity.
It seems odd to report on the biochemical properties of R315C but determine the structure of R315A.Line 283."occludes access to FAD via Tyr361" There isn't a Tyr361 is the sequence.Figure 6.Is it Tyr361 or Tyr403?(how conserved is this residue?)Line 312 and elsewhere, is it Tyr361 or Tyr403?Line 319."abolished catalytic activity and retention of the FAD cofactor observed with cMTHFRR315A".This was not experimentally shown.
Line 382: "phosphorylation status and effector-induced and stabilized conformational changes,", remove the second "and" Supporting information Line 92.Complete the following sentence: Patients with 1081C>T have 92 low MTHFR activities, 5~27% of controls; thus, most of them are in severe (usually the activity range is 0%-20% of controls MTHFR deficiency9,10 93 .
Line 99: Please specify the mutant in the following sentence: "Therefore, it is likely that the mutant undergoes a post-translational modification, such as phosphorylation

Manuscript Number: NCOMMS-23-59767
Response to reviewers (Referee comments in black; responses in red) Referee 1 1.Initially, I was somewhat misled by the section on the human enzyme because the introduction emphasizes the fungal protein.Please add a couple of sentences about the project's logic, explaining why it started with the human enzyme and its pathological mutants.
We appreciate the reviewer's comments and therefore have included a couple of sentences in the introduction to state that the overarching goal was to understand the allosteric regulation of MTHFR biochemically and structurally.Initially, the human protein was studied and while extremely valuable biochemical data was collected, it proved recalcitrant to structural studies, leading us to pivot to a eukaryotic MTHFR model in the fungal protein.The introduction has been edited to include the specific rationale for starting with the human MTHFR and why the fungal protein MTHFR homolog/model system was ultimately focused on.
2. The text and experiments about phosphorylation (lines 102-130) should be mostly removed as phosphorylation is not addressed in this manuscript.Move it to the Supplementary Information (SI).The main text should simply mention the authors' attempt to crystallize the human enzyme and the discovery of new phosphorylation sites.
We agree with the reviewer's comment and have moved this section (lines 102-107) to the Supplementary Information (SI Figs. 1 and 2. Identification of phosphorylation sites of recombinant wild-type hMTHFR) to emphasize the discovery of the three new phosphorylation sites.We have condensed the following section (lines 123-130) to highlight the importance of the biochemical data obtained from the human MTHFR and added references to rationalize the use and study of specific patient mutations.Additionally, a plot containing summarized biochemical data for patient mutations has been added to the Supplementary Information (SI Fig. 4).
3. Line 131 should explicitly state that the Arg357Cys mutation was chosen based on published data.
We agree with the reviewer's comment and have added the relevant reference in the main text, along with a brief sentence summarizing the specific mutation: "The Arg357Cys patient mutant is based on rare mutation (1081C>T, Arg357Cys)" (Goyette in Am.J. Hum. Genet. 59, 1268-1275(1996).With regards to providing steady-state kinetic parameters, the assay used (NADPH:menadione oxidoreductase assay) is not under steady-state conditions.Additionally, and more importantly, MTHFR is an allosteric enzyme, and thus does not obey standard Michaelis-Menten kinetics.
While we are actively engaged in a more detailed kinetic analysis of this enzyme, we believe its discussion falls outside the scope of this paper.The main goal behind Figure 2 was to establish the fungal protein's suitability as a biochemical model for eukaryotic MTHFRs.Our preliminary data suggest that the mechanism of AdoMet inhibition, particularly the binding kinetics in solution for cMTHFR, will align with those previously conducted by Jencks and Matthews (JBC 1986).5.In Figure 3, the legend should indicate which ligands are bound and the coloring used to depict them.
An explicit mention of the ligands bound to each structure and their respective colors have been included, and the Figure 3 legend has been revised to explicitly mention that the R-state cMTHFR structure does not contain AdoHcy bound.
6. Lines 200-215: Move the limited proteolysis data text after presenting the 3D structure.Use it as an experiment validating the relevance in solution of the described conformational changes.
We agree with the reviewer's suggestion and have moved this section after Figure 3 is introduced and edited the text to emphasize how the limited proteolysis data provides a biochemical validation of the conformational dynamics in solution.
7. Line 230: The biochemical properties of Arg315Ala should be minimally described (activity, spectra).Spectra for the R315A mutant have been added to Fig. 2d.There is no discernable difference between the R315C and R315A mutants, spectroscopically or biochemically with regards to their activity (R315C: 14% relative to wild-type, R315A: ~14.7% relative to wild-type).Notably, the R315A mutant displays the same FAD absorbance quench and red-shift at 450 nm, altering the absorbance maxima of FAD from 453 nm to 463 nm.
8. The methods indicate that R-state (not the T-state?) crystallisation was done with a triple E21Q, L393M, V516F mutant where the lysines were vhemically methylated.Please explain this in the main text.
We have added a couple of sentences in the main text stating the use of the triple mutant: L393M and V516F were introduced to disrupt AdoMet binding, as previous work with the human enzyme has shown that AdoMet was copurified during protein purification (Froese et al. (2018)), which could explain why no AdoHcy was found bound in our R-state structure; E21Q mutant was introduced in an attempt to co-crystallize cMTHFR in the R-state with CH3H4-folate, but we were not successful.Additionally, a rationale is also provided behind the use of reductive lysine methylation to aid in crystallization, along with relevant references regarding the use of this method to enhance the crystallizability of proteins.
The relevant text has been edited to highlight that the structures of individual MTHFR domains as compared via superposition of (regulatory vs. regulatory, catalytic vs. catalytic) between the R and T-states demonstrates that they are structurally rigid and maintain the same topology; in other words, the regulatory domain of the R and T-state superimpose with an RMSD of 0.53 Å and the catalytic domains with an RMSD of 0.39 Å.This indicates that flexibility and conformational dynamics are not mediated by MTHFR domain(s) restructuring, but by rearrangement of the linker connecting the domains.10. Figure 5 is confusing due to a likely mistake in residue numbering.For instance, the legend states that Arg357 interacts with Glu360, whereas the figures outline an interaction between Arg358 and Glu360.Thr368 is not sohwin in the figure .We appreciate the reviewer pointing this out and have fixed the residue numbering mistakes and included the outlined interactions in Figure 5.The figure legend has been changed to reflect the residue numbering of the fungal enzyme.
11. Related to the above point: The manuscript is sometimes challenging to follow due to the backand-forth between the residue numbering of the human and fungal enzyme (e.g., lines 290-300).I recommend using the residue numbering of the fungal enzyme for consistency.
We appreciate the reviewer pointing out this source of confusion and have revised the main text to use the residue numbering of the fungal enzyme for consistency.The residue numbering corresponding to the human enzymes has been left solely for the results section regarding the biochemical characterization of human MTHFR and for Figure 8 and the model presented for the human MTHFR allosteric regulation (this could be changed).

Referee 2
1.The poor atomic resolution of both the R-and T-state of the cMTHFR structures is a limitation of the study.The resolution is suitable for tracking the reorganization of domain, but perhaps not for mapping intermolecular interactions.Can you show an H-bond between R358 and Glu360 at 3.4 A resolution?
We value and acknowledge the reviewer's comments regarding the limitations of mapping intermolecular H-bonding interactions given the resolution of our R-state structure.While the figure in question (Figure 5) shows the human R-state structure (6FCX) which has a higherresolution (2.50 Å), we have changed the figure to reflect our R-state structure and residue numbering for clarity and consistency.
In addition, we have edited the text and figure to emphasize our focus on the distribution of charge of the residues, particularly those surrounding the linker and R315.This focus is crucial as the R315A mutant has proven sufficient for capturing and elucidating the structure of the elusive Tstate.A comparison between the human R-State structure and our R-state structure has been included in the Supplementary Information (SI Fig. 12).
2. Biochemical data supporting the binding of two equivalent of SAM would confirm that the second binding site is physiologically relevant or at least happens in solution.Can this be done with ITC?Given that site 2 is occluded in the R state and site 1 is not, they would presumably have different binding affinities.You could measure SAM binding to wild type cMTHFR and the R315 mutant.
We greatly value the reviewer's comments highlighting the unusual stoichiometry observed for AdoMet/SAM binding.For over a year, we have dedicated extensive efforts to determine binding affinities for AdoMet using both the wild-type enzyme and the R315 mutant, employing native (gas-phase) mass spectrometry (IM-MS) and conducted equilibrium binding measurements using ITC, as recommended by reviewer 2. However, these efforts have not yet provided conclusive data.However, despite these efforts, conclusive data remains elusive at this time Using IM-MS, the wildtype and R315A mutant were found to be highly heterogenous, and individual contributions between states in the presence and absence of ligand could not be deconvoluted.In our hands, the best sample and runs confirmed that the wildtype is found as a dimer, with two FADs bound.
Froese et al. 2018 demonstrated using native MS that as-purified enzyme has AdoMet bound: their N-terminal truncated construct, which lacks the phosphorylation site/region showed up to two AdoHcy bound, but they attributed this to AdoMet degradation; full-length hMTHFR showed up to 2 AdoMet molecules bound, but upon phosphatase treatment, they saw 2 AdoHcy molecules.
They argue that this was the result of AdoMet degradation to AdoHcy.Given that cMTHFR lacks the N-terminal phosphorylation region, it is possible that AdoMet could likewise be degraded.
During the submission of our work, we came across a preprint (Froese et al., bioRxiv, 2024) of a study published this month in Nat.Comm.by the same group that investigated human MTHFR and its allosteric regulation, leading to the discovery of the first human MTHFR T-state structure.
They obtained results that complement and validate ours, finding that two molecules of AdoMet are bound in their T-state structure.In their new study, the authors were able to conduct ITC experiments with the human enzyme and determined that the binding stoichiometry of AdoMet was double that of AdoHcy, indicating that the 2:1 stoichiometry observed in the T-state structure is not an artifact.However, while they were able to provide Kd values, the binding affinities of each site could not be determined separately.Additionally, the question of cooperativity was not addressed.
Jencks and Mathews (JBC, 1986) determined/observed the spectrophotometric titration of AdoMet to MTHFR and tracked the subsequent absorbance quench of FAD as a function of AdoMet concentration, finding that the resulting curve was sigmoidal/biphasic in nature and posited: "The two phases could be due to either fast AdoMet binding to the R state followed by a slower isomerization to, e.g.AdoMet-ligated T state, or binding of first one and then a second AdoMet to R state enzyme.Our model adopts the first of these explanations." Our initial AdoMet titration to cMTHFR wild-type in Fig. 2c was fit using a Michaelis-Menten/hyperbolic curve; however, fitting the same data using a sigmoidal curve/Hill plot gave a superior fit (R 2 = 0.9831 vs. R 2 = 0.9568), with Hill coefficient equal to 2.09, and an IC50 of 18.78 µM; the sigmoidal nature of the FAD quench and the positive Hill coefficient suggests that AdoMet binding is cooperative, and that there are thus multiple AdoMet binding events, the first of which facilitates the binding of the second AdoMet molecule.The "slow" step observed and first reported by Jencks and Mathews is thus best explained by the second of their two hypotheses: that there are two AdoMet binding events, and that the binding of the first AdoMet molecule triggers the R-T state transition by cooperatively enabling the binding of a second AdoMet molecule.The observed positive cooperativity validates our structural analysis, since binding of the first AdoMet is necessary to reveal the cryptic secondary AdoMet site.
To determine AdoMet binding stoichiometry, Jencks and Matthews (1986) performed spectrophotometric titration of AdoMet and tracked Abs450-503 vs. [AdoMet].They showed that their data is best fit by a theoretical model "computed for a model with two AdoMet-binding sites, where AdoMet binding to R or T state subunits produces independent effects on the spectrum." Our data mimics this model: Absorbance changes were monitored at 503 and 450 nm, with complete spectra also recorded.The absorbance changes were similar to those reported previously (Jencks and Matthews, 1986): "a decrease in the flavin absorbance at 450 nm, an increase in the long wavelength shoulder of the 450 nm band (maximal at 503 nm) and an isosbestic point at 485 nm".The best-fit curve is a 4-parameter logistic (4PL) model, with a Hill coefficient of 2.318 and IC50 of 19.72 (>99% confidence, Akaike's information criterion implemented in Prism, R 2 : 0.9934).95% confidence intervals are shown as dashed lines.
The only other enzyme that binds two molecules of AdoMet for allostery is, to our knowledge, Arabidopsis Thaliana threonine synthase, where AdoMet acts as an allosteric activator.Using a similar approach, Curien et al. (Biochemistry, 1998) showed spectrophotometrically that AdoMet binding was cooperative, with a Hill coefficient of 2: "Since such a value represents the maximal value that can be obtained for a dimeric enzyme both subunits are strongly constrained by their association, suggesting that they change their conformation upon SAM binding in a concerted manner." As such, while we have been unable to obtain binding affinities for AdoMet to either the wild-type or the R315 mutant, we believe that the spectrophotometric data indicate that the observation of two molecules of AdoMet in our T-state structure is not an artifact, and that we do indeed observe multiple AdoMet binding events in solution.Froese et al.'s results confirm our findings, and together, they complement each other.Although we are currently working on discerning the specific binding affinities of each AdoMet event, we feel that further delving into this aspect is outside the purview of this paper.We appreciate the reviewer for catching this mistake.Reference 21 was removed and in its place, Fig 2a.Why was free FAD added?Why did it slow the initial velocity of the reaction?Provide more information in the figure legend for 2a and in Results section.Catalytic preference for NADPH vs NADH should be a ratio of their kcat/Km values.

Figure 5 .
Figure5.The labelling of the residues appears off.Is Arg358 supposed to be Arg357 and is Arg357 supposed to Arg 377 in the zoomed in image?
4. InFigure 2 and associated text, provide steady-state kcat and KM parameters for the fungal enzyme.The Figure 2 legend should list the enzyme and substrate concentrations used to obtain the curves and spectra shown.Specific values for the concentrations of cMTHFR used and relevant substrate concentrations have been added to the figure legend (50 nM protein for panels a, b, and d) (100 μM of NADPH or NADH for panel a; for additions in panel a, 100 μM NADPH combined with 2 μM FAD or 100 μM AdoMet) (10 μM protein for panel c along with 0-200 μM AdoMet for panel c).The methods have been lightly edited to fix a transcription error regarding the amount of protein used to obtain the data in panel c (fixed to 10 μM).

Figure 1 .
Figure 1.Spectrophotometric titration of cMTHFRwt with AdoMet.UV-Vis spectral changes of cMTHFR wt (10 µM) titrated with varying concentrations of AdoMet (0-200 µM).Absorbance changes were monitored at 503 and 450 nm, with complete spectra also recorded.The absorbance changes were similar to those reported previously (Jencks and Matthews, 1986): "a decrease in the flavin absorbance at 450 nm, an increase in the long wavelength shoulder of the 450 nm band (maximal at 503 nm) and an isosbestic point at 485 nm".The best-fit curve is a 4-parameter logistic (4PL) model, with a Hill coefficient of 2.318 and IC50 of 19.72 (>99% confidence, Akaike's information criterion implemented in Prism, R 2 : 0.9934).95% confidence intervals are shown as dashed lines.

3.
Line 63, Fig 1B What is the % of sequence identity between the cMTHFR and human MTHFR.Does the 38% just compare the catalytic domain and regulatory domains, without the linker?We appreciate the reviewer pointing out this source of confusion.The values only refer to the domains, not the linker.The figure has been revised for clarity.4. Line 154.Reference 21 did not appear measure hMTHFR catalytic preference for NADPH versus NADH.